Abstract

Background

Androgen insensitivity syndrome (AIS) comprises a range of phenotypes from male infertility
to complete feminization. Most individuals with AIS carry germline mutations of the
androgen receptor (AR) that interfere with or ablate its function. As genital fibroblasts
retain expression of the AR in vitro, we used genital skin fibroblasts from normal males and 46,XY females with complete
AIS due to known AR mutations to gain insights into the role of the AR in human genital differentiation.

Results

Using DNA microarrays representing 32,968 different genes, we identified 404 transcripts
with significant differences in transcription levels between genital skin fibroblasts
cultured from normal and AIS-affected individuals. Gene-cluster analyses uncovered
coordinated expression of genes involved in key processes of morphogenesis. On the
basis of animal studies and human genetic syndromes, several of these genes are known
to have specific roles in genital differentiation. Remarkably, genital fibroblasts
from both normal and AIS-affected individuals showed no transcriptional response to
dihydrotestosterone treatment despite expression of the AR.

Conclusions

The results suggest that in addition to differences in the anatomic origin of the
cells, androgen signaling during prenatal development contributes to setting long-lasting,
androgen-independent transcriptional programs in genital fibroblasts. Our findings
have broad implications in understanding the establishment and the stability of sexual
dimorphism in human genital development.

Background

Development of the male genitalia is largely controlled by cells in the urogenital
mesenchyme that express androgen receptors (AR) [1,2]. Germline mutations of the AR gene produce a spectrum of developmental abnormalities in 46,XY individuals ranging
from infertility or mild hypospadias to complete feminization, which are collectively
referred to as the androgen insensitivity syndrome (AIS). In general, the degree of
genital ambiguity correlates with the level of compromise of AR function: 46,XY individuals
with AR-inactivating mutations are completely feminized despite high levels of serum
testosterone [3-5].

The molecular events responsible for AR-dependent male genital morphogenesis are poorly
understood. We hypothesized that the AR-dependent mesenchymal programs underlying
male external genitalia development might be illuminated by comparing the transcriptional
profile of mesenchyme-derived stromal cells from normal males to those from individuals
affected with AIS. As cultured genital fibroblasts originate from the urogenital mesenchyme
and retain expression of the AR in vitro, we compared gene-expression patterns in cultured genital fibroblasts from normal
46,XY males and from 46,XY females with severe or complete AIS, using DNA microarrays
representing 32,968 distinct human genes. We found striking differences in the gene
expression profiles of genital fibroblasts cultured from normal and AIS patients,
but no transcriptional response to androgen was detectable in any of the cultured
genital fibroblasts.

Results

Baseline gene transcription in genital fibroblasts

To gain insights into the role of androgen in genital morphogenesis, we compared basal
transcriptional patterns in genital fibroblasts from 46,XY individuals with either
wild-type AR or germline inactivation of the AR as a result of mutation. We could
not identify mutations in the AR gene in two phenotypically female individuals with complete AIS. However, genital
skin fibroblasts of both subjects failed to express AR protein and did not show androgen
binding (Table 1). Initially, we restricted our analysis to genital skin fibroblasts grown from the
foreskin of nine normal males and from the labia majora of five AIS-affected, 46,XY
individuals with female external genitalia. The AR status of all genital fibroblasts
was confirmed by AR gene sequencing and hormone-binding assays (Table 1). To determine basal gene-expression patterns, mRNA was isolated from growth-arrested
(G0) confluent cells and analyzed using DNA microarrays of approximately 43,000 cDNAs
representing 32,968 genes. Distinct differences in the basal expression profiles of
normal and AIS-derived fibroblasts allowed these two groups to be distinguished on
the basis of their expression patterns by unsupervised hierarchical clustering analysis
(Figure 1). We then identified 404 unique transcripts (represented by 487 total cDNAs) with
significant differences in expression levels between normal and AIS genital fibroblasts
using the significance analysis of microarrays (SAM)-procedure [6], with a false discovery rate of less than 0.92% (percent of genes identified by chance
alone).

We used the list of the 487 cDNAs from the SAM analysis for hierarchical clustering
analysis of 24 different primary fibroblast lines from normal and AIS affected individuals
(Figure 2). In addition to the 14 genital-skin fibroblast lines used to generate the SAM list,
we included five gonadal fibroblast lines from 46,XY females with complete AIS, a
prostate fibroblast cell line from a normal male (analyzed twice), abdominal skin
fibroblasts from a normal male, forearm fibroblasts from a normal male, and genital
fibroblasts from two AIS 46,XY females who had documented AR mosaicism (ARD364, ARD
465, Table 1). The ARD364 and ARD465 lines express wild-type AR and were derived from individuals
who were mosaics for wild-type AR and AR with a premature stop codon [7]. Hierarchical cluster analysis, based on the expression of 472 of the 487 previously
identified transcripts with measurable expression across at least 80% of 24 experiments,
separated the fibroblasts into those with gene-expression patterns resembling normal
male foreskin fibroblasts and a second group with an expression pattern more similar
to labial skin fibroblasts from AIS-affected individuals (Figure 2). The latter group contained all five gonadal fibroblast lines from complete AIS
females as well as the fibroblast lines from abdominal and forearm skin. The prostate
fibroblasts, on the other hand, displayed expression patterns largely similar to the
normal male foreskin cells. Notably, the two mosaic AIS cell lines ARD364 and ARD465
showed gene-expression patterns that most resembled normal male foreskin (Figure 2).

Figure 2. Hierarchical cluster analysis of genes and experiments based on cDNAs identified as
being significantly different in expression between normal genital skin fibroblasts
and genital skin fibroblasts of female patients with AIS. The left panel shows an
overview of 472 of the total of 487 significant transcripts that showed measurable
expression across at least 80% of 24 experiments. The color code of the dendrogram
and the sample names represent the origin of the fibroblast strains. GSF, genital
skin fibroblast; PRF, prostate fibroblast; GOF, gonadal fibroblast; FSF, forearm skin
fibroblast; ASF, abdominal skin fibroblast; other abbreviations are as in Figure 1 and Table 1. The scale ranges from -4 to +4 in log2 space. For the complete dataset, see Additional data files and [36].

Comparison of expression patterns in genital fibroblasts from normal and AIS-affected
individuals, and fibroblasts from extragenital sites, offers possible insights into
the programs that underlie genital development. For instance, transcripts encoding
homeobox A13 protein (HOXA13) and T-box proteins (TBX) showed striking differences
in their expression levels between the 'male genital' and 'AIS/extragenital' fibroblasts.
HOXA13 was expressed at high levels in normal male foreskin fibroblasts and at low levels
in all AIS and extragenital fibroblasts (Figure 2). T-box gene 3 (TBX3) was expressed at higher levels in the fibroblasts from genital skin, extragenital
skin or prostate from males than in genital skin fibroblasts from AIS 46,XY females
(Figure 2). TBX2 showed an almost identical expression profile to TBX3, whereas high TBX5 expression appeared to be restricted to foreskin fibroblasts from normal males (including
those from the phenotypically female mosaic patient ARD364). BMP4 (bone morphogenetic protein 4) was predominantly expressed in foreskin fibroblasts
from normal males and in prostate fibroblasts (Figure 2). WNT2 (wingless-type MMTV integration site family member 2) was part of a small gene cluster
with high expression in normal male foreskin fibroblasts that distinguished these
samples from all other fibroblasts (Figure 2).

We tested the responses of normal and AIS genital fibroblasts to dihydrotestosterone
(DHT), under culture conditions similar to those that were reported to produce aromatase
induction in these cells [8]. Cells were treated with DHT (100-1,000 nM) both at confluency (G0) and during exponential growth, and transcript levels were assessed using DNA microarrays.
Unsupervised hierarchical clustering did not disclose any obvious differences in gene-expression
patterns between DHT-treated and ethanol-treated fibroblasts, either in normal controls
or in AIS-derived cell lines. We treated LNCaP prostate cancer cells with androgen
under similar conditions and readily identified nearly 500 transcripts modulated by
androgen with unsupervised hierarchical clustering analysis and with supervised methods
([9] and data not shown). A supervised analysis comparing gene-expression patterns of
DHT-treated fibroblasts to ethanol-treated controls was carried out using the SAM
procedure. Again, no genes could be identified that were significantly induced or
repressed by DHT treatment. Additional experiments using physiological concentrations
of androgen (for example, 0.01 - 1 nM methyltrienolone) also failed to disclose any
androgen-responsive genes (data not shown). In contrast, SAM analysis identified 1,664
transcripts that differed significantly between proliferating and confluent cells,
and 1,232 transcripts that differed between fibroblasts derived from AIS-affected
individuals and normal male foreskin. Hierarchical cluster analysis of these experiments
clearly showed the distinct differences in transcriptional profiles between AIS-derived
and normal male fibroblasts and between proliferating and normal fibroblasts (Figure
3). Cells derived from the same individual and cultured under the same conditions always
showed highly similar gene-expression patterns, suggesting that the differences in
expression between individuals are stable and reproducible despite passage in vitro (Figure 3).

Figure 3. Hierarchical cluster analysis of genes and experiments with different DHT-treatment
regimens. Shown are 686 transcripts whose log2 red/green ratio differed from the mean expression level across all experiments by
at least 1.5 in at least three experiments. The analysis is based on 2,862 transcripts
identified by SAM analysis that distinguish between normal genital skin fibroblasts
and gonadal fibroblasts from 46,XY female AIS patients, and between proliferating
and confluent fibroblasts. The color code in the dendrogram depicts the origin of
the fibroblast cultures. The gray and white bars at the top of the cluster indicate
the proliferation state of the samples. On the right, the regions of the cluster diagram
that differentiate between normal and AIS-derived fibroblasts, and proliferating and
confluent cells, respectively, are indicated. No differences in transcript levels
could be discerned between DHT-treated and control cells in either normal foreskin
fibroblasts or fibroblasts from AIS-affected 46,XY females. Abbreviations are as in
Figure 2 and Table 1. The scale ranges from -8 to +8 in log2 space. For the dataset of 686 genes used for the cluster analysis and the complete
dataset of 2,862 genes, see Additional data files and [36].

Discussion

We found consistent, characteristic differences in baseline gene expression patterns
between genital skin fibroblasts from normal males and 46,XY female patients with
AIS. Many of these differences between normal and AIS-derived fibroblasts were also
observed in gonadal fibroblasts, suggesting that these differences are not purely
due to differences in the anatomical site of origin of the fibroblasts. Interestingly,
fibroblasts derived from abdominal and forearm skin, regions with relatively little
sexual dimorphism, showed gene-expression patterns similar to the labial skin fibroblasts
of AIS patients. Together, these data suggest that the AR is involved in determining
a stable and stereotypical program of gene expression in genital fibroblasts that
does not require the continuing presence of androgen for its maintenance.

A critical question raised by these results is whether the observed differences between
genital fibroblasts from males and AIS females reflect cell-autonomous effects of
androgen exposure during development, or indirect effects of the AR-dependent genital
morphogenetic program. One possible interpretation of these data is that the distinct
patterns of expression could have been due to differences in the origin or the developmental
milieu of foreskin fibroblasts, derived from the genital tubercle, as opposed to the
labial fibroblasts, derived from the genital swellings [10]. The differences in gene expression we observed in AIS fibroblasts of gonadal origin
compared to those of labial origin support this view (Figure 3). We have observed consistent and characteristic differences in the gene-expression
patterns of skin fibroblasts derived from different locations on the body [11]. However, the current set of experiments suggests that the androgen receptor has
a cell-autonomous role which contributes to a stable androgen-independent gene-expression
pattern in genital fibroblasts. Expression patterns in cultured labial skin fibroblasts
derived from two different individuals with AR mosaicism suggested that the cell-autonomous
AR status was a relevant determinant of baseline gene expression in genital skin fibroblasts.
Both these fibroblast lines, although derived from morphologically female genitalia
in phenotypically female 46,XY individuals mosaic for AR-inactivating mutations, expressed
wild-type AR in the cultured cells. These female AIS-affected individuals are thought
to have acquired their AR gene mutations post-zygotically [12]. ARD364, which showed AR protein expression and binding in the range of normal male
foreskin fibroblasts ([7] and see Table 1), despite its origin from anatomically female genitalia, had a gene-expression pattern
indistinguishable from foreskin fibroblasts of normal males (Figure 2). The second fibroblast line from an AR mosaic patient, ARD465 (Table 1), had very low wild-type AR expression and showed baseline gene-expression patterns
that were nevertheless more similar to normal male foreskin and prostate fibroblasts
than to any of the AIS-derived cell lines (Figure 2).

The discrepancy between the female phenotype of these mosaic individuals despite expression
of the wild-type AR in cultured genital skin fibroblasts is not resolved to date [7]. It may be explained by a time-dependent rise of an originally small fraction of
cells containing the wild-type AR allele in the mosaic genital mesenchyme during prenatal and postnatal development,
or by differences between in vivo and in vitro conditions. Yet, the documented expression of the wild-type AR in cultures of these
labial cells supports the idea that the AR status of the fibroblast was an important
intrinsic determinant of the basal transcription patterns we identified. Therefore,
the AR appears to be involved in setting long-lasting gene-expression patterns in
genital skin fibroblasts.

Comparison of gene-expression patterns in genital fibroblasts from normal and AIS-affected
individuals, and in fibroblasts from extragenital sites, may offer clues to the programs
that underlie external genital development. Both cell adhesion and connective tissue
remodeling are indispensable for normal development and maintenance of tissue integrity
[13-15]. The differential expression of proteoglycans, collagens and cell adhesion molecules
(for example cadherin 13) might be involved in genital morphogenesis and later stability
of sexually dimorphic traits of the external genitalia. Some genes expressed in wild-type
AR cells could influence androgen signaling. For instance, aldo-keto reductase 1C1
is specifically involved in cellular androgen metabolism [16] and thus may modulate the spectrum of cellular androgenic steroids available for
activation of the AR. Structurally different androgens elicit different patterns of
response from several androgen-responsive promoters, suggesting that the type of ligand
present could affect cellular response [17]. Mitogen-activated protein kinase 14, and STAT-induced STAT inhibitors 2 and 3 were
expressed at significantly higher levels in cells with wild-type AR. Both MAP kinase
and STAT pathways are involved in AR-dependent regulation and in ligand-independent
activation of the AR [18]. Differential expression of aldehyde dehydrogenase 1A1 and alcohol dehydrogenase
1B, enzymes that affect retinoic acid biosynthesis, suggest that other signaling pathways
may participate in the AR-initiated programs of external genital differentiation [19,20].

Several genes expressed specifically in the normal male foreskin fibroblasts have
been previously implicated in male genital development, including HOXA13, the T-box genes, BMP4 and DWnt2. Mutations in HOXA13 can cause distal limb and urogenital-tract malformations such as male hypospadias
in hand-foot-genital syndrome [21]. T-box genes (TBX) are essential early regulators of limb development and also appear to be involved
in male genital development [22,23]. Mutations in TBX3 cause the ulnar-mammary syndrome characterized by limb, apocrine, and genital developmental
abnormalities [23]. Expression of T-box genes 2, 3, and 5 was significantly higher in normal male foreskin
fibroblasts than in AIS genital fibroblasts. BMP4 has been implicated in ductal budding and branching during prostate development [24] and a potential role of BMP4 in external genital development has also been postulated
[25]. DWnt2 has been found to have roles in sex-specific cell determination in the gonads and
genital disc of Drosophila [26]. Thus, mutations in genes characteristically expressed in normal male foreskin fibroblasts
can, in some cases, lead to defective genital development. The data from these experiments
therefore provide candidate genes for further investigation in patients with genital
malformations.

As normal genital skin fibroblasts of 46,XY male individuals express the AR in vitro (see Table 1 and [4,7]), we had anticipated that androgen treatment would elicit a transcriptional response
program that could provide additional insights into the role of androgen in genital
development. We have previously used a similar approach to delineate the transcriptional
programs activated in prostate cancer cells in response to androgen [9]. We had hoped that comparison of transcriptional responses of normal fibroblasts
to those from AIS-affected individuals with varying degrees of genital ambiguity would
provide still further insights into androgen's role in genital morphogenesis. However,
we were unable to detect any significant changes in gene-expression patterns in cultured,
AR-expressing genital fibroblasts or in AIS-derived fibroblasts in response to androgens.
Although two previous reports have shown increases in aromatase enzymatic activity
in genital skin fibroblasts treated with dihydrotestosterone (DHT) [8,27], others have failed to observe changes in aromatase activity in response to androgen
[28]. In agreement with our findings, Elmlinger et al. found significantly different baseline expression levels of insulin-like growth
factor (IGF) and insulin-like growth factor binding protein (IGFBP) between normal
and AIS-derived genital skin fibroblasts, and could not detect changes in transcript
levels in response to androgen treatment [29]. In normal genital fibroblasts, androgen-responsive reporter genes can only be activated
by expression of co-transfected AR in the presence of ligand [30]. Therefore, endogenous AR expression itself may be insufficient in genital skin fibroblasts
to elicit a transcriptional response. Moreover, the lack of detectable changes in
transcript levels for any of the 30,000 genes in the AIS-fibroblasts virtually excludes
the possibility that DHT or R1881 could be acting through other steroid receptors
or other signaling pathways.

The differences in androgen responsiveness we have observed between normal genital
fibroblasts and prostate cancer epithelial cells in vitro might reflect the responses seen in vivo. Prostate epithelial cells retain exquisite sensitivity to androgen throughout life.
Androgen deprivation produces profound involution of the prostate, particularly of
the epithelial component, but little or no change in the external genitalia. It is
possible that genital mesenchymal cells are only capable of responding to androgen
at discrete stages in development in their specific in vivo environment. In mice, stromal androgen responsiveness is restricted to the earliest
stages in prostate development, and later the epithelial compartment becomes responsive
and remains so [1]. This responsiveness may be mediated through the expression of specific AR co-regulators.
Compared to LNCaP cells, normal male genital fibroblasts show distinctly lower baseline
expression of several AR co-regulators (such as NCOA2 (GRIP-1), NCOA3 (TRAM-1), ARA54
(RNF14), data not shown). Thus, genital fibroblasts may express critical AR co-regulators
at discrete times during development that allow them to respond by setting up long-lasting
transcriptional programs that underlie the genesis and maintenance of genital morphology.

Conclusions

Our data suggest that in addition to androgen-independent positional influences on
fibroblast phenotypes, the AR is originally involved in establishing stable and reproducible
patterns of gene expression in stromal cells during genital differentiation, which
are reflected in the differences in global gene-expression patterns between fibroblasts
cultured from the genital skin of normal individuals and females affected by AIS.
Comparison of the expression patterns of genital fibroblasts from 46,XY normal males
and 46,XY females with inactivated AR provides a window on the AR-dependent gene-expression
programs within the urogenital mesenchyme, which contribute to the development and
structural integrity of male and female genitalia. For further discrimination of androgen-independent
positional effects from prenatal androgen actions on expression phenotypes of genital
fibroblast strains, comparative expression profiling of homologous genital tissues
is needed. The apparent lack of response of genital fibroblasts to androgen in vitro, despite expression of a normal AR, has important implications for future research
in defining the role of androgen in genital development and the pathogenesis of ambiguous
genitalia. Transcriptional profiling of the early stages of genital development in vivo in the presence and absence of androgen may provide further insights into the role
of androgen in genital development.

Materials and methods

The study was approved by the ethical committee of the University of Lübeck, Germany.
Informed consent was obtained from all normal subjects and AIS patients or their parents.

Cell strains

Primary cultures of genital fibroblasts were established from genital skin biopsies
(labia majora) or gonadal biopsies in female AIS patients and from the foreskin of
normal males undergoing circumcision. Abdominal skin fibroblasts were derived from
the midline above the mons pubis of a fertile male during abdominal surgery. Forearm
skin fibroblasts from a normal male were a gift from H. Chang (Department of Biochemistry,
Stanford University). Peripheral zone prostate fibroblasts were a gift from D. Peehl
(Department of Urology, Stanford University) and were established from a histologically
normal region of a patient undergoing prostatectomy for prostate cancer who had not
been previously treated with hormonal therapy. Hormone-binding assays using methyltrienolone
(R1881, 17β-hydroxy-17α-methyl-4,9,11-estrotrien-3-one) and androgen receptor sequencing
have been described previously [7].

Cell culture and hormone treatment

For determination of basal gene-expression profiles without androgen stimulation,
fibroblasts were cultured on 150-mm plastic dishes at 37°C with 5% CO2. To eliminate possible artifacts due to differing states of proliferation, cells
were grown to confluence, at which point they enter G0 arrest [31]. They were maintained in phenol-red-free DMEM F12 (Dulbecco's modified Eagle Medium
with the nutrient mix F12; Gibco) containing L-glutamine, 15 mM Hepes buffer, penicillin/streptomycin (Gibco) and 12.9% of a constant
lot of certified fetal calf serum (FCS; Gibco). The pH was adjusted to 7.4 with 1
N NaOH and the medium was exchanged every 48 h. At day 13 the last media exchange
was carried out and 96 h later cells were scraped and mRNA harvested directly.

Androgen stimulation of genital fibroblasts was carried out under two different culture
conditions similar to those previously reported to produce induction of aromatase
enzymatic activity in these cell lines [8,27]. In the first, cells were grown to confluence as described above using phenol-red-free
DMEM F12 containing L-glutamine, 15 mM Hepes buffer, penicillin/streptomycin (Gibco) with 12.9% charcoal-stripped,
steroid-free FCS (D/S-FCS) (Hyclone) to ensure androgen-depleted conditions in control
cells. With every media exchange every 48 h, cells received either ethanol in a final
dilution of 1:100,000 or 100 nM dihydrotestosterone (DHT) dissolved in ethanol. The
last DHT treatment was administered with the last media exchange 96 h before lysate
preparation. In total, six doses of either ethanol or 100 nM DHT were given.

In the second set of experiments, cells were cultured to confluence for 14 days as
described above. They were then trypsinized and seeded at a density of 3,000 cells
per cm2 in 150-mm plates. Twenty-four hours later, medium was removed, and cells were washed
three times with new media containing 12.9% D/S-FCS, then cultured for another 24-h
interval in the absence of androgens. Cells were then treated with either 1:100,000
ethanol, 100 nM or 1,000 nM DHT dissolved in ethanol. After 24 h incubation, exponentially
growing cells were harvested. LNCaP cells, passaged and treated under similar conditions,
were used as a positive control for androgen reponsiveness.

RNA isolation and cDNA labeling

Protocols for mRNA preparation and cDNA labeling are available online [32]. mRNA (2 μg) from single experiments was reverse transcribed and labeled with Cy5
(pseudo-coloured red) and pooled reference mRNA was labeled with Cy3 (pseudo-coloured
green). Reference mRNA contained equal mixtures of fibroblast mRNA (pooled from confluent
and proliferating cultures of normal and AIS genital skin fibroblasts) and a 'common
reference' of mRNAs isolated from 11 different proliferating cultured tumor cell lines
that we have described previously [33].

Microarrays and hybridizations

Microarrays with approximately 43,000 sequence-validated PCR-amplified human cDNAs
representing 32,968 UniGene clusters were manufactured as described [32]. Hybridizations were performed using equal amounts of Cy3- and Cy5-labeled cDNAs
according to previously published protocols [32]. Hybridized microarrays were scanned using a GenePix4000 array scanner and analyzed
with GenePix Pro 3.0 software (Axon Instruments, Union City, CA).

Microarray data analysis

Only spots with fluorescence signals 1.5-fold greater than background in either the
experimental or reference samples were included in the analysis. To correct for variations
in cDNA labeling efficiency, we normalized the Cy5/Cy3 fluorescence ratios for all
genes in each array hybridization to obtain an average log2 (ratio) of 0. We restricted our analysis to genes with measurable expression in 80%
of the samples we analyzed. We used the SAM procedure [6] to identify genes with statistically significant differences in baseline expression
levels between normal and AIS genital fibroblasts. The SAM procedure computes a two-sample
T-statistic (for example for normal vs AIS cell lines) for the normalized log ratios
of gene-expression levels for each gene. It thresholds the T-statistics to provide
a 'significant' gene list and provides an estimate of the false discovery rate (the
percent of genes identified by chance alone) from randomly permuted data. Gene-expression
data were clustered [34,35] and results were visualized using TreeView software [35].

To identify the effects of androgen treatment on gene expression in genital fibroblasts,
we carried out a set of 21 DNA microarray analyses of mRNA from normal and AIS genital
fibroblasts. This dataset included cells treated at confluence (G0) or during exponential proliferation as described above. Raw data were filtered,
normalized and centered as described above. We used the SAM procedure to identify
transcripts with significant differences in expression with reference to the origin
of the fibroblast lines, whether the cells were confluent or proliferating, and whether
they had been treated with androgen.

Additional data files

The following files are available: a figure (Additional data file 1) showing the complete dataset for Figure 1, with associated array tree correlations
(atr), complete data table (cdt) and gene tree correlations (gtr) files (Additional
data files 2, 3 and 4); a figure (Additional data file 5) showing the complete dataset for Figure 2, with associated atr, cdt and gtr files
(Additional data files 6, 7 and 8); a figure (Additional data file 9) showing the complete dataset for Figure 3, with associated atr, cdt and gtr files
(Additional data files 10, 11 and 12). Figure 3 contains 686 transcripts whose log2 red/green ratio differed from the mean expression level across all experiments by
at least 1.5 in at least three experiments of the treatment series. The analysis was
based on 2,862 transcripts that differed significantly between proliferating and confluent
cells and between fibroblasts derived from AIS-affected individuals and normal male
foreskin, respectively, as identified by SAM analysis of the treatment series. The
complete 2,862 genes are displayed in two further figures (Additional data files 13 and 14) with associated atr, cdt and gtr files (Additional data files 15, 16 and 17). All files are also available online at [36].

Additional data file 5. Hierarchical cluster analysis of genes and experiments based on cDNAs identified as
being significantly different in expression between normal genital skin fibroblasts
and genital skin fibroblasts of female patients with AIS. The left panel shows an
overview of 472 of the total of 487 significant transcripts that showed measurable
expression across at least 80% of 24 ex-periments. The color code of the dendrogram
and the sample names represent the origin of the fibroblast strains. The scale ranges
from -4 to +4 in log2 space. (Complete dataset for Figure 2.)

Additional data file 9. Hierarchical cluster analysis of genes and experiments with different DHT treatment
regimens. Shown are the 2862 transcripts that distinguish between normal genital skin
fibroblasts and gonadal fibroblasts from 46, XY female AIS patients, and between proliferating
and confluent fibroblasts. The color code in the dendrogram depicts the origin of
the fibroblast cultures. The gray and white bars on top of the cluster indicate the
proliferation state of the samples. On the right, the regions of the cluster diagram
are indicated which differentiate between normal and AIS-derived fibroblasts, and
proliferating and confluent cells, respectively. No differences in transcript levels
could be discerned between DHT treated and control cells in either normal foreskin
fibroblasts or fibroblasts from AIS affected 46, XY females. The scale ranges from
-8 to +8 in log2 space. (Complete dataset for Figure 3.)

Additional data file 13. Upper half of Figure. Hierarchical cluster analysis of genes and experiments with
different DHT treatment regimens. Shown are the 2862 transcripts that distinguish
between normal genital skin fibroblasts and gonadal fibroblasts from 46, XY female
AIS patients, and between proliferating and confluent fibroblasts. The color code
in the dendrogram depicts the origin of the fibroblast cultures. The gray and white
bars on top of the cluster indicate the proliferation state of the samples. On the
right, the regions of the cluster diagram are indicated which differentiate between
normal and AIS-derived fibroblasts, and proliferating and confluent cells, respectively.
No differences in transcript levels could be discerned between DHT treated and control
cells in either normal foreskin fibroblasts or fibroblasts from AIS affected 46, XY
females. The scale ranges from -8 to +8 in log2 space. Complete dataset from which
Figure 3 was created.

Additional data file 14. Lower half of Figure. Hierarchical cluster analysis of genes and experiments with
different DHT treatment regimens. Shown are the 2862 transcripts that distinguish
between normal genital skin fibroblasts and gonadal fibroblasts from 46, XY female
AIS patients, and between proliferating and confluent fibroblasts. The color code
in the dendrogram depicts the origin of the fibroblast cultures. The gray and white
bars on top of the cluster indicate the proliferation state of the samples. On the
right, the regions of the cluster diagram are indicated which differentiate between
normal and AIS-derived fibroblasts, and proliferating and confluent cells, respectively.
No differences in transcript levels could be discerned between DHT treated and control
cells in either normal foreskin fibroblasts or fibroblasts from AIS affected 46, XY
females. The scale ranges from -8 to +8 in log2 space. Complete dataset from which
Figure 3. was created.